P693: DIAGNOSTIC ROLE OF CD26+ LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA
Authors: S. Tiwari, J. Dass, G. Vishwanathan, R. Dhawan, M. Agarwal, P. Kumar, T. Seth, S. Tyagi, M. Mahapatra
Journal: HemaSphere (2022)

Background: Diagnosis of CML consists of persistent leucocytosis with the demonstration of the Philadelphia (Ph) chromosome abnormality, the t(9;22)(q34;q11), by routine cytogenetics, fluorescence in situ hybridization (FISH) or by molecular studies such as RT-PCR.These conventional methods suffer from a longer turn around time, higher costs and requirement of labs with adequate expertise. CD26 is a highly specific marker expressed in CML stem cells, which is not present on normal hematopoietic stem cells or on Leukemia stem cells (LSCs) of other myeloid neoplasms. A flow-cytometry based assessment of CD26+LSCs may prove to be an optimal biomarker for the diagnosis and monitoring of CML patients. Aims: 1)Assessment of CD 26 expression in suspected cases of CML-chronic phase 2)Correlation of CD 26+ stem cells with clinico-pathological parameters at baseline and its kinetics on tyrosine kinase inhibitor (TKI) treatment on further follow-up at 12 months. Methods: Suspected patients of CML-CP were included in the study. Peripheral blood were utilized for flowcytometric assessment of CD26+ LSCs. The results were correlated with conventional diagnostic tests of CML. Patients with myeloid neoplasms other than CML who proved negative for BCR-ABL1 by RT-PCR and normal HSCs donors treated with granulocyte-colony stimulating factors (G-CSF), were included as negative controls. All samples were processed using standard stain -lyse-wash method within 24 hr of collection. To reach a sensitivity of 10−5, acquisition of at least 1.0 × 106 cells was performed on FACS Canto II flow cytometer data a FACS DIVA 8.0.3 software (BD, Biosciences).CD26 expression was evaluated by a sequential gating strategy. After exclusion of doublets and debris CD45 dim to intermediate/CD34+/CD38- population was gated sequentially and then the expression of CD 26+ was studied on this population. All the samples were simultaneously assessed for BCR-ABL1 transcript by standard RT-PCR analysis. Patients on treatment were also followed up at 12 months to detect levels of CD26+ LSCs and correlated with BCR -ABL1 levels by qPCR. Results: A total of 86 patients of suspected cases of CML were included in the study from June 2020 to Dec 2021.The On flowcytometry analysis, CD 26 expression was present in 75 cases. All the cases with positive CD26+ LSCs were found to be positive on RT-PCR for BCR-ABL transcript. Eleven cases which were scored negative for CD26+ LSCs were also found to be negative for any BCR-ABL transcript. These negative cases included 8 cases of primary myelofibrosis, 2 cases of chronic myelomonocytic leukemia and 1 case of Polycythaemia Vera. The median percentage of CD26+ LSCs was 39.8% (Range-0.5-100). There was no significant correlation of the CD26 levels with age (p value=0.96), sex (p value=0.7), percentage of blasts (p value = 0.88), total leucocyte counts (p value=0.32), basophils (p value = 0.19), Sokal score (p value = 0.99) and ELTS score (p value = 0.99). Follow-up data was available for 7 patients and at 12 months all of these patients had achieved major molecular remission on TKIs. Two patients scored negative for CD26+LSCs while five patients showed significant fall in the level of CD 26+ LSCs however it was still detectable by flowcytometry (p value= 0.04). Summary/Conclusion: Flowcytometry assessment of CD26 + LSCs is a rapid and cheap diagnostic tool for diagnosis of CML with high diagnostic efficacy. Falling levels on TKIs warrants further research to prove it as a surrogate for monitoring response by conventional methods.